Faculty Bibliography

Akt1 is implicated in cell metabolism, survival migration, and gene expression; however, little is known about the role of specific Akt isoforms during inflammation in vivo. Thus, we directly explored the roles of the isoforms Akt1 and Akt2 in acute inflammation models by using mice deficient in either Akt1 or Akt2. Akt1(-/-) mice showed a markedly reduced edema versus Akt2(-/-) and WT controls, and the reduced inflammation was associated with a dramatic decrease in neutrophil and monocyte infiltration. The loss of Akt1 did not affect leukocyte functions in vitro, and bone marrow transplant experiments suggest that host Akt1 regulates leukocyte emigration into inflamed tissues. Moreover, carrageenan-induced edema and the direct propermeability actions of bradykinin and histamine were reduced dramatically in Akt1(-/-) versus WT mice. These findings are supported by in vitro experiments showing that Akt1 deficiency or blockade of nitric oxide synthase markedly reduces histamine-stimulated changes in transendothelial electrical resistance of microvascular endothelial cells. Collectively, these results suggest that Akt1 is necessary for acute inflammation and exerts its actions primarily via regulation of vascular permeability, leading to edema and leukocyte extravasation

Endosymbioses have dramatically altered eukaryotic life, but are thought to have negligibly affected prokaryotic evolution. Here, by analysing the flows of protein families, I present evidence that the double-membrane, gram-negative prokaryotes were formed as the result of a symbiosis between an ancient actinobacterium and an ancient clostridium. The resulting taxon has been extraordinarily successful, and has profoundly altered the evolution of life by providing endosymbionts necessary for the emergence of eukaryotes and by generating Earth's oxygen atmosphere. Their double-membrane architecture and the observed genome flows into them suggest a common evolutionary mechanism for their origin: an endosymbiosis between a clostridium and actinobacterium.

BACKGROUND: Hairy/Enhancer of Split (Hes) proteins are targets of the Notch signaling pathway and make up a class of basic helix-loop-helix (bHLH) proteins that function to repress transcription. Data from Hes1 deficient mice suggested that Hes1, like Notch1, is necessary for the progression of early T cell progenitors. Constitutive activation of Notch is known to cause T cell leukemia or lymphoma but whether Hes1 has any oncogenic activity is not known. METHODOLOGY/PRINCIPAL FINDINGS: We generated mice carrying a Hes1 transgene under control of the proximal promote of the lck gene. Hes1 expression led to a reduction in numbers of total thymocytes, concomitant with the increased percentage and number of immature CD8+ (ISP) T cells and sustained CD25 expression in CD4+CD8+ double positive (DP) thymocytes. Hes1 transgenic mice develop thymic lymphomas at about 20 weeks of age with a low penetrance. However, expression of Hes1 significantly shortens the latency of T cell lymphoma developed in Id1 transgenic mice, where the function of bHLH E proteins is inhibited. Interestingly, Hes1 increased expression of a subset of Notch target genes in pre-malignant ISP and DP thymocytes, which include Notch1, Notch3 and c-myc, thus suggesting a possible mechanism for lymphomagenesis. CONCLUSIONS/SIGNIFICANCE: We have demonstrated for the first time that Hes1 potentiates T cell lymphomagenesis, by up-regulating a subset of Notch target genes and by causing an accumulation of ISP thymocytes particularly vulnerable to oncogenic transformation.

A rooted tree of life provides a framework to answer central questions about the evolution of life. Here we review progress on rooting the tree of life and introduce a new root of life obtained through the analysis of indels, insertions and deletions, found within paralogous gene sets. Through the analysis of indels in eight paralogous gene sets, the root is localized to the branch between the clade consisting of the Actinobacteria and the double-membrane (Gram-negative) prokaryotes and one consisting of the archaebacteria and the firmicutes. This root provides a new perspective on the habitats of early life, including the evolution of methanogenesis, membranes and hyperthermophily, and the speciation of major prokaryotic taxa. Our analyses exclude methanogenesis as a primitive metabolism, in contrast to previous findings. They parsimoniously imply that the ether archaebacterial lipids are not primitive and that the cenancestral prokaryotic population consisted of organisms enclosed by a single, ester-linked lipid membrane, covered by a peptidoglycan layer. These results explain the similarities previously noted by others between the lipid synthesis pathways in eubacteria and archaebacteria. The new root also implies that the last common ancestor was not hyperthermophilic, although moderate thermophily cannot be excluded.

Diabetes is associated with poor outcomes following acute vascular occlusive events. This results in part from a failure to form adequate compensatory microvasculature in response to ischemia. Since vascular endothelial growth factor (VEGF) is an essential mediator of neovascularization, we examined whether hypoxic up-regulation of VEGF was impaired in diabetes. Both fibroblasts isolated from type 2 diabetic patients, and normal fibroblasts exposed chronically to high glucose, were defective in their capacity to up-regulate VEGF in response to hypoxia. In vivo, diabetic animals demonstrated an impaired ability to increase VEGF production in response to soft tissue ischemia. This resulted from a high glucose-induced decrease in transactivation by the transcription factor hypoxia-inducible factor-1alpha (HIF-1alpha), which mediates hypoxia-stimulated VEGF expression. Decreased HIF-1alpha functional activity was specifically caused by impaired HIF-1alpha binding to the coactivator p300. We identify covalent modification of p300 by the dicarbonyl metabolite methylglyoxal as being responsible for this decreased association. Administration of deferoxamine abrogated methylglyoxal conjugation, normalizing both HIF-1alpha/p300 interaction and transactivation by HIF-1alpha. In diabetic mice, deferoxamine promoted neovascularization and enhanced wound healing. These findings define molecular defects that underlie impaired VEGF production in diabetic tissues and offer a promising direction for therapeutic intervention.

Telomeres are thought to be maintained by the preferential recruitment of telomerase to the shortest telomeres. The extension of the G-rich telomeric strand by telomerase is also believed to be coordinated with the complementary synthesis of the C strand by the conventional replication machinery. However, we show that under telomere length-maintenance conditions in cancer cells, human telomerase extends most chromosome ends during each S phase and is not preferentially recruited to the shortest telomeres. Telomerase rapidly extends the G-rich strand following telomere replication but fill-in of the C strand is delayed into late S phase. This late C-strand fill-in is not executed by conventional Okazaki fragment synthesis but by a mechanism using a series of small incremental steps. These findings highlight differences between telomerase actions during steady state versus nonequilibrium conditions and reveal steps in the human telomere maintenance pathway that may provide additional targets for the development of anti-telomerase therapeutics

OBJECTIVES: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. METHODS: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites-both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates-were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. RESULTS: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for approximately 12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. CONCLUSIONS: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.

Signal transduction along the Ras/MAPK pathway has been generally thought to take place at the plasma membrane. It is now evident that the plasma membrane is not the only platform capable of Ras/MAPK signal induction. Fusion of Ras with green fluorescent protein and the development of genetically encoded fluorescent probes for Ras activation have revealed signaling events on a variety of intracellular membranes including endosomes, the Golgi apparatus and the endoplasmic reticulum. Thus, the Ras/MAPK pathway is spatially compartmentalized within cells and this may afford greater complexity of signal output